Aminoglycosides are a family of highly potent, broad-spectrum antibiotics which can be used to treat infections caused by gram-negative as well as gram-positive bacteria. However, these antbiotics have a narrow therapeutic index and are potentially nephrotoxic and ototoxic. (M. Jolley, et al., Clin. Chem. 27(7), 1190-1197 (1981); U.S. Pat. No. 5,079,234) Consequently, it is desirable for patients treated with aminoglycosides to be under close clinical observation to guide the safe therapeutic use of these antibiotics. An effective tool in the guidance is the monitoring of aminoglycoside concentration in the biological fluid of a treated patient, and there is a continuing effort to improve the performance and sensitivity of assays for aminoglycoside antibiotics.
In testing for analytes such as drug molecules (including aminoglycosides), immunoassays, particularly competitive binding immunoassays, have proven to be especially advantageous. In competitive binding immunoassays, an analyte in a biological sample competes with a labeled reagent, also known as an analyte analog or tracer, for a limited number of receptor binding sites on antibodies specific for the analyte and analyte analog. Enzymes such as xcex2-galactosidase and peroxidase, fluorescent molecules such as fluorescein compounds, radioactive compounds such as 125I, and microparticles are common labeling substances used in tracers. The concentration of analyte in the sample determines the amount of analyte analog which will bind to the antibody. The amount of analyte analog that will bind is inversely proportional to the concentration of analyte in the sample, because the analyte and the analyte analog each bind to the antibody in proportion to their respective concentrations. The amount of free or bound analyte analog can then be determined by methods appropriate to the particular label being used.
The modification of aminoglycosides for use as well defined tracers in immunoassays can be problematic. The synthesis of aminoglycoside tracers typically yields a mixture of products, rather than site specific derivatives, due to the presence of multiple amino (Nxe2x80x94H) functionalities (U.S. Pat. Nos. 5,849,599; 4,816,391; 4,328,311; Mallams, J.C.S. Perkins 1, 1981, 2186-2208.). The desired product is difficult to separate from the mixture, and cannot typically be characterized as a single product. This uncertainty leads to poor reproducibility of the synthesis and consequently to variations in product quality. Also, any further manipulations or modifications of the product are difficult, since the presence of unwanted products (side products) may interfere with the desired product. Variations in product quality can cause the analysis to have different characteristics for different batches of the tracer, thus requiring the user to calibrate the analysis each time a different batch of tracer is used.
It is thus desirable to provide aminoglycoside derivatives which can be easily isolated as pure single products. It is also desirable to be able to modify the aminoglycosides to increase their utility under a wider variety of assay conditions.
In one aspect of the invention, there is a method of making a derivatized aminoglycoside, comprising reacting an aminoglycoside of formula (I) with at least 2 equivalents of a divalent metal ion in an aprotic solvent to complex two neighboring amino group and hydroxyl group pairs; 
wherein A is xe2x80x94CH2NH2, xe2x80x94CHCH3NH2, or xe2x80x94CHCH3NHCH3; B is xe2x80x94H or xe2x80x94OH; D is xe2x80x94H or xe2x80x94OH; E is xe2x80x94NH2 or xe2x80x94OH; G is xe2x80x94NH2, or xe2x80x94NHCH3; J is xe2x80x94H or xe2x80x94OH; L is xe2x80x94H, xe2x80x94CH3, or xe2x80x94OH; Q is xe2x80x94H or xe2x80x94CH2OH; and Y is xe2x80x94H, or xe2x80x94C(xe2x95x90O)CH(OH)CH2CH2NH2; reacting the non-complexed amino groups with a protecting reagent to provide protecting groups; removing the divalent metal ion to provide two unprotected amino groups; reacting one of the unprotected amino groups with a reactive substance comprising -T; and removing the protecting groups to produce a compound of formula (II) 
wherein Gxe2x80x2 is xe2x80x94NH2, xe2x80x94NHCH3, xe2x80x94NH-T, or xe2x80x94NCH3-T; Yxe2x80x2 is xe2x80x94H, xe2x80x94C(xe2x95x90O)CH(OH)CH2CH2NH2, or -T; and T is a linker group, a carrier, or a label.
In another embodiment of the invention, there is a reagent for an assay, comprising a compound of formula (II) 
wherein A is xe2x80x94CH2NH2, xe2x80x94CHCH3NH2, or xe2x80x94CHCH3NHCH3; B is xe2x80x94H or xe2x80x94OH; D is xe2x80x94H or xe2x80x94OH; E is xe2x80x94NH2 or xe2x80x94OH; Gxe2x80x2 is xe2x80x94NH2, xe2x80x94NHCH3, xe2x80x94NH-T, or xe2x80x94NCH3-T; J is xe2x80x94H or xe2x80x94OH; L is xe2x80x94H, xe2x80x94CH3, or xe2x80x94OH; Q is xe2x80x94H or xe2x80x94CH2OH; and Yxe2x80x2 is xe2x80x94H, xe2x80x94C(xe2x95x90O)CH(OH)CH2CH2NH2, or -T; T is a linker group, a carrier, or a label; and -T is present in only one of Gxe2x80x2 or Yxe2x80x2; and wherein the purity of the compound is at least 90%.
In yet another embodiment of the invention, there is an antibody produced in response to the above reagent.
In yet another embodiment of the invention, there is an assay method for determining an aminoglycoside, comprising combining a sample suspected of containing said aminoglycoside with an antibody specific for said aminoglycoside and with the above reagent, the reagent comprising the analyte analog of said aminoglycoside, and the reagent capable of forming a detectable complex with said antibody; and determining the presence or amount of said detectable complex as a measure of said analyte in said sample.
In yet another embodiment of the invention, there is an assay method for determining an aminoglycoside, comprising combining a sample suspected of containing said aminoglycoside with a reagent and with the above antibody, the reagent comprising an analyte analog of said aminoglycoside, and the reagent capable of forming a detectable complex with said antibody; and determining the presence or amount of said detectable complex as a measure of said analyte in said sample.
In yet another embodiment of the invention, there is a compound of formula (II): 
wherein A is xe2x80x94CH2NH2, xe2x80x94CHCH3NH2, or xe2x80x94CHCH3NHCH3; B is xe2x80x94H or xe2x80x94OH; D is xe2x80x94H or xe2x80x94OH; E is xe2x80x94NH2 or xe2x80x94OH; Gxe2x80x2 is xe2x80x94NH2, xe2x80x94NHCH3, xe2x80x94NHxe2x80x94X, or xe2x80x94NCH3xe2x80x94X; J is xe2x80x94H or xe2x80x94OH: L is xe2x80x94H, xe2x80x94CH3, or xe2x80x94OH; Q is xe2x80x94H or xe2x80x94CH2OH; and Yxe2x80x2 is xe2x80x94H, xe2x80x94C(xe2x95x90O)CH(OH)CH2CH2NH2, or xe2x80x94X; and wherein xe2x80x94X is present in only one of Gxe2x80x2 or Yxe2x80x2 and is a moiety of formula (VI II), (IX), or (X) 
In yet another embodiment, there is a test kit comprising the above reagent, the above compound, or the above antibody.